With over 50,000 identified members, terpenoids comprise the largest known class of natural products. These compounds are structurally diverse, although based on related carbon skeletons. The structural diversity found among these compounds allows them to perform a variety of essential biochemical functions. These compounds serve as attractants for pollinators, antimicrobial and antiherbivorial defense compounds, and may react with reactive oxygen species to protect against oxidative damage (Dudereva et al., Plant Physiol. 135:1893-1902 (2004)). As components of the essential oils of aromatic plants, they are largely responsible for the distinct flavors and fragrances associated with their host plants. Moreover, the value of these small molecules extends beyond their biological utility. Many terpenoids have commercial value as antibiotics, pest control agents, fragrances, flavors, and anti-cancer agents, among other important uses.
A specific class of these natural products, sesquiterpenoids, is derived from a common 15-carbon building block. This common 15-carbon building block is farnesyl pyrophosphate (FPP). Many important products, such as the flavoring nootkatone, the cosmetic additive bisabolol, and amorpha-4,11-diene, a precursor to the antimalarial compound artemisinin, are sesquiterpenoids and thus are based on the 15-carbon skeleton of FPP. Therefore, methods that can increase the yield of FPP that can be utilized in sesquiterpenoid synthesis are of extreme importance.
To maximize production of terpenes, mutations in squalene synthase have been used to prevent or minimize conversion of farnesyl pyrophosphate to squalene. In practice, this has been done by either eliminating the corresponding gene, reducing its expression using weak promoters, or controlling its expression with a regulated promoter. Squalene is a precursor to sterols, which are essential to viability of yeast and other organisms. Accordingly, complete elimination of the gene requires feeding of sterols. The yeast Saccharomyces cerevisiae is not normally capable of taking up sterols under aerobic conditions, so in order to feed sterols to mutants, secondary mutations enabling sterol uptake are required.
Various solutions have been proposed in order to obtain high yields of farnesyl pyrophosphate for maximum production of terpenes. In one approach, the ERG9 gene of the yeast is completely eliminated. The gene ERG9 encodes the enzyme squalene synthase. However, because these mutants in which ERG9 is eliminated cannot synthesize squalene, which is a precursor to sterols, they must be fed sterols (Takahashi, et al., Biotech. Bioengineer. 97:170-181 (2007)). In another approach, PCT Patent Application Publication No. WO 06/102342 by Bailey et al., describes production of high yields of farnesyl pyrophosphate by modifying the expression or activity of one or more polypeptides involved in generating cytosolic acetyl-CoA and/or NADPH. In another approach, a promoter, the MET3 promoter, is used in place of the native ERG9 promoter to downregulate the expression of squalene synthase by repressing its synthesis by adding methionine, which acts as a repressor with respect to the MET3 promoter (Asadollahi et al., Biotech. Bioengineer. 99:666-677 (2007)). In a similar approach, in addition to repression of ERG9 production, overproduction of a soluble, truncated form of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and enhancement of the activity of the transcription factor UPC2 was employed (Paradise et al., Cell. Metabol. Engineer. Bioengineer. 100:371-378 (2008); Ro et al., Nature 440: 940-943 (2006)).
However, there is a need for improved strains of Saccharomyces cerevisiae that can overproduce FPP without the need for sterol supplementation and without regulating expression. Preferably, these improved strains would grow efficiently and produce high levels of farnesyl pyrophosphate for subsequent terpenoid synthesis.